Two separate categories of pharmacological vaccine adjuvants: phosphodiesterase (PDE) inhibitors and inducible nitric oxide synthase (iNOS) inhibitors when given concurrently with immunization enhance immune memory. The iNOS protein is a key mediator of T cell death following activation, and its inhibition during an immune response increases the number of surviving T cells, leading to enhanced memory. Prolonged signaling via cAMP achieved by PDE4 inhibition also blocks a death pathway. We propose to examine questions crucial to the further development of these pharmacological vaccine adjuvants. The first question addresses the molecular basis of the connection between cAMP-mediated signaling and induction of iNOS in responding CD4 T cells. We will test the hypothesis that enhanced signaling through cAMP-dependent protein kinase A (PKA) suppresses the induction of transcription factor or factors critical for iNOS expression. This will be done by stimulating purified CD4 T cells in vitro through CD3 and CD28 in the presence or absence of pharmacological agents that inhibit PDE or PDE4, or agents that enhance cAMP signaling by other pathways, with or without PKA inhibitors. We will then measure the induction of iNOS mRNA as well as protein expression by RT-PCR and Western blot analyses, accompanied by the analysis of induction of known critical transcription factors for iNOS, namely, NF-kB and IRF-1. In the second question, which addresses the mechanisms and the utility of these agents, we will ask if these agents enhance persistence of immune memory in aged animals, where T cell memory responses are compromised. We will examine the effect of the administration of these agents on the generation of T cell memory in young and aged mice in immunization systems both in vitro and in vivo. Inhibition of iNOS leads to enhanced T cell memory survival by inhibition of neglect-induced death of T cells post- activation. However, responding naive T cells from aged mice also undergo death during primary activation, associated with generation of effector memory cells rather than central memory cells. We will test the hypothesis that this early death pathway in naive T cells from aged mice is distinct from the post-activation pathways of death, and that therefore neither PDE nor iNOS inhibition will affect either this death pathway or the effector to central memory ratio in aged mice. We will purify naive CD4 T cells from aged mice and activate them in vitro through CD3 and CD28 to assess proliferation, maturation and death in the presence or absence of PDE or iNOS inhibitors. This work is the US portion of an Indo-US Vaccine Action Plan program. RELEVANCE STATEMENT: Long lasting vaccination memory that is effective in both healthy and at-risk individuals is an important public health goal. This work seeks to improve the quality and persistence of such immune memory by adding drugs that decrease the death of immune cells during a first encounter with a vaccine. This work will define the sorts of vaccines that these pharmacological interventions can be useful with, their potential utility in the compromised immune responses of elderly individuals, and their biochemical mechanisms of action.